The present invention relates to nucleic acid sequences from Bacillus thuringiensis, and, in particular, to genomic DNA sequences that encode insecticidal proteins from B. thuringiensis strains EG5858, EG4332 and EG4096. In addition, proteins and fragments thereof and antibodies capable of binding specifically to the proteins and methods of using the disclosed nucleic acid molecules, proteins, protein fragments and antibodies are encompassed by the present invention.
The gram-positive bacterium Bacillus thuringiensis is well known as a source of insecticidal proteins, most of which accumulate in crystalline inclusions during sporulation (Crickmore et al., Microbiol. Mol. Biol. Rev. 62:807-813, 1998; de Maagd, et al., Annu. Rev. Genet. 37:409-433, 2003; Schnepf et al., Microbiol. Mol. Biol. Rev. 62:775-806, 1998). These crystal proteins, or Cry proteins, are diverse and include distinct protein families as judged by amino acid sequence analysis and structural information derived from X-ray crystallography. A comprehensive listing of known BT insecticidal proteins, genes, and information about the nomenclature associated with these genes and proteins can be accessed at the web site biols.susx.ac.uk/home/Neil_Crickmore/Bt/index.htmL. A variety of crystalline Bt insecticidal proteins are known. These proteins collectively display a wide range of activity against insect pests, including those from the orders Lepidoptera, Coleoptera, Diptera, Hemiptera, Hymenoptera, and Heteroptera. In addition to the delta-endotoxin crystal proteins, B. thuringiensis strains also produce secreted insecticidal proteins that are expressed during vegetative growth, including VIP1-, Vip2-, and Vip3-type proteins, TIC901 and related proteins, and TIC900 and related proteins.
The use of Cry proteins in agriculture for insect control has been advanced by the development of transgenic crops. Transgenic crops expressing B. thuringiensis cry genes have enjoyed an unprecedented rate of adoption by farmers in the United States and elsewhere since their introduction in the 1990s. Worldwide acreage of all transgenic crops in 2002 was reported to be approximately 58.7 million hectares, or 145 million acres, representing a 12% increase over year 2001 acreage (James C., ISAAA Briefs No. 27. ISAAA, Ithaca, N.Y., 2002). The long term success of this transgenic approach to insect control will depend on appropriate insect resistance management (IRM) strategies. An important component of a sustainable IRM strategy is the discovery and deployment of new insecticidal proteins with distinct modes-of-action that either prevent or significantly delay the development of widespread resistance among insect pest populations.
Other bacteria besides B. thuringiensis display insecticidal properties that may in part be attributed to insecticidal proteins. The gram-negative bacteria Photorhabdus luminescens, Xenorhabdus nematophilus and related species, Serratia entomophila, Pseudomonas syringae, and Yersinia pestis all express insecticidal proteins. Each of these species have also been shown to produce a large extracellular multi-protein complex consisting primarily of TC proteins (toxin complex). Each of the proteins within the complex individually have generally failed to display insecticidal activity (ffrench-Constant and Bowen, Curr. Opin. Microbiol. 2:284-2880, 1999; ffrench-Constant et al., FEMS Microbiol. Rev. 759:1-24, 2002; Waterfield et al., Trends Microbiol. 9:185-191, 2001). In addition to the tc-like genes, other insect virulence genes such as the mcf genes have been discovered (Waterfield, et al., FEMS Microbiol. Lett. 229:265-270, 2003). The tc homologs and mcf genes could be a valuable source of insect-resistance traits for future transgenic crops because they are unrelated to the B. thuringiensis cry genes and therefore likely exhibit different modes of action. Recently, genes encoding proteins that exhibited similarity to the TC proteins were also identified in a gram-positive bacterium identified as a Paenibacillus species (US Patent Application Publication No. 2004/0110184). Paenibacillus bacteria have been determined to be phylogenetically unrelated with any other bacteria including other aerobic, endospore-forming bacilli based upon rRNA and phenotypic characteristics (Ash et al., Antonie Van Leeuwenhoek 64:253-260, 1993).
There is a continuing need to identify additional genes that encode insecticidal proteins useful in producing insect resistant transgenic plants. With few exceptions, Bt crystalline insecticidal proteins all seem to exhibit a similar structural and functional motif. The proteins generally exhibit a conserved three-dimensional structure based on x-ray crystallographic studies of several members of the Bt delta-endotoxin family. Generally, the proteins also exhibit a similar mode of action in that they all bind in a specific fashion to membranes surfaces within the midgut of the larval form of a particular target insect species and subsequently form ion-channel pores that result in a disruption of the membrane potential along the surfaces of the membrane, which generally results in the death or incapacitation of the larval form of the target insect. Transgenic plants expressing these proteins have been demonstrated to be effective in minimizing insect infestation, however, there is a concern that insect populations repeatedly exposed to these insect resistant plants could develop resistance to the Bt toxin produced within the plants. Therefore, there is a need to identify additional insecticidal proteins that are different from the typical Bt insecticidal crystal protein and that exert their insecticidal effects through a mode of action that is also different from the typical Bt insecticidal crystal protein.
Bacillus thuringiensis strains are not known that exhibit insecticidal activity against a diversity of insect targets. Generally, B. thuringiensis strains exhibit insecticidal activity against one or at most two orders of insect species, such as for example, against Dipteran and Lepidopteran insect species. The inventors herein have identified three strains of B. thuringiensis that surprisingly exhibit insecticidal activity against a diversity of insect orders including Lepidoptera, Coleoptera, Diptera, and Hemiptera. Bt insecticidal proteins are known to be expressed from genes located on extrachromosomal megadalton plasmids. It was believed that such broad insecticidal diversity exhibited by these strains was a result of expression of one or more toxin genes located within one or more megadalton plasmids in each of these strains. The inventors herein have identified an operon within one of these strains that contains genes encoding proteins that exhibit similarity to the gram-negative TC proteins. In addition, the genes within the B. thuringiensis operon substantially maintain the order of expression of the gram-negative genes described above.